A single-tube nucleotide isolation reagent for the quantitative PCR detection of virus in body fluids

Abstract

A high-salt reagent composed of guanidinium thiocyanate, guanidine hydrochloride, urea, sodium citrate, and other compounds was designed for the single-tube isolation of viral nucleotides from body fluids. The single-tube reagent was used for the extraction of SIV RNA and HBV DNA from standard virus stock dilutions and virus-positive samples. The sensitivity and reproducibility of the single-tube reagent were analysed via quantitative PCR assays. The results revealed that the single-tube reagent can facilitate quantitative PCR-mediated detection in a reaction system with a 25-μl volume using only 100μl of a body fluid sample and reaches a sensitivity of up to 50copies/ml. The low coefficients of variance of both the HBV and SIV standard stock results indicate the excellent reproducibility of the single-tube reagent. A Bland–Altman analysis of the assay results from the SIV- and HBV-positive samples revealed that the single-tube reagent can precisely extract both RNA and DNA viral nucleotides from virus-positive samples. All of the isolation steps were performed in a single tube and were completed in no more than 35min. The only major equipment required is a high-speed freezing centrifuge. The single-tube reagent is economical and easy to use and does not require any complex equipment.